Phosthetic structures derived from collagen

ABSTRACT

Artificial ivory and bone-like structures are formed from a complex partial salt of collagen with a metal hydroxide and with an ionizable acid such as calcium hydroxide and phosphoric acid. Calcium phosphate may be intimately mixed with the complex partial collagen salt before shaping into a desired configuration. Structural strength may be increased by incorporating fibers and other ions may be included to increase hardness.

United States Patent 1191 Cruz, Jr.

[ Oct. 23, 1973 PHOSTHETIC STRUCTURES DERIVED FROM COLLAGEN [75]Inventor: Mamerto M. Cruz, Jr., Pennington,

[73] Assignee: Avicon, lnc., Ft. Worth, Tex.

[22] Filed: Oct. 20, 1971 21 Am No.: 190,817

Related US. Application Data [63] Continuation-in-part of Ser. No.823,959, May 12,

1969, abandoned.

[52] US. Cl. 106/161, 128/1 [51] C08h 7/06, C08h 17/04 [58] Field ofSearch l06/l6l, l 55 [56] References Cited UNITED STATES PATENTS3,443,261 5/1969 Battista iO6/l6l 11/1968 Talty etal l06/l55 4/1960Highberger 106/155 Primary Examiner-Theodore Morris Att0rneyGeorge F.Mueller et al.

[57] ABSTRACT 10 Claims, No Drawings PHOSTHETIC STRUCTURES DERIVED FROMj COLLAGEN This application is a continuation-in-part of my copendingapplication Ser..No. 823,959, filed May 12, 1969, now abandoned.

This invention relates to a new composition of matter, particularly wellsuited for prosthetic devices, and to a process for manufacturing thesame.

The supporting skeleton of vertebras consists of cartilage and bone. Inthe embryonic stages, cartilage forms this supporting skeleton and mostof the cartilage is replaced in the adultby bone. Cartilage persists inadults at bone joints and in other locations such as the ear, nose, etc.The principal organic constituent of cartilage and bone tissue iscollagen and the principal inorganic component in cartilage and bonetissue is calcium phosphate complexes or compounds such as, for example,hydroxyapatite. The chief inorganic constitutents are calcium,magnesium, phosphate radicals, carbonate radicals, the fluoride radicaland water, the compounds being of varying compositions generallybelonging to the apatite group. Other inorganic ions are generallypresent in trace amounts and other organic matterv is also present. Theother elements found in minor and trace amounts in bone tissue arealuminum, barium, boron, chlorine, copper, iron, lead, manganese,potassium, sodium, strontium and tin while arsenic bismuth, lithium,molybdenum,- nickel, selenium, silicon, silver and "zinc have beendetected spectrographically. In general, the differences in hardness andrigidity between cartilage and bone tissue are due to the differences incomposition such as differences in the ratios of collagen 'totheinorganic calcium phosphate compounds and the presence-of other radicalsas'well as the morphological structure. 1

The bone tissue consists of bundles of collagenous fibers in anamorphous cement material which is probably a protein-polysaccharidecomplex impregnated with the calcium phosphate complexes or compounds.Sulfur is also included and appears to be present as ester sulfateassociated with the polysaccharides. Differencesin hardness and othercharacteristics of the different bone tissues and parts of bone arebelieved to be due to variations in amounts of calcium carbonate andother constituentssuch as magnesium fluorine, the carbonate radical,etc. absorbed on the surface of the hydroxyapatite crystals, or thefluoride and carbonate radical may replace the hydroxy radical.Regardless of the precise manner in which these additional substancesare associated with the calcium phosphate compounds, these substancesappear to alter the crystal lattices of the phosphate compounds.Differences in physical characteristics are also influenced by themorphology of the specific or individual bone tissue. Roughly, the ratioof the collagenous material'to the inorganic material in the human boneis slightly less than 1:3, varying from about 1:2.65 to about 1:2.89 inthe human femur, based on the weightof dry defatted bone. The foregoingis obviously an oversimplification of the structure and composition ofbone tissue which in fact is a most complex structure varying in precisecomposition with age, individual and species of mammals.

The precise method by which this class of tissue is formed is notknown..Physico-chemical theories have been advanced in an attempt toexplain the formation of dental calculusand it is possible that bonetissue might be formed in a somewhat similar manner. According to thistheory, saliva is considered to be a colloidal solution of proteinswhich is more or less saturated with calcium and phosphate ions. Surfacetension is believed to cause the proteins to concentrate at the surfaceof the saliva thus reducing the viscosity of the liquid and causing asettling out of inorganic salts which deposit on the teeth surfaces.

Thus far, cartilage and bone tissue has not beenformed or duplicatedsynthetically. ln bone surgery, a variety of materials has been usedincluding bone, bone derivatives and synthetic substitutes. Bone fromwhich certain constituents such as minerals, proteins, lipids and waterhave been removed is generally classed as bone derivative. Syntheticsubstitutes include metals, certain synthetic polymers, calcium sulfateand hydroxyapatite. V

The chemistry of sparingly soluble phosphate salts or specifically thesystem Ca-P O -2H,O and the precise chemistry and structure of thecalcium phosphate compounds occurring in natural cartilage and bonetissue are extremely complex. Accordingly, the term calcium phosphate isused herein and in the claims to include dicalcium phosphate, tricalciumphosphate, octacalcium phosphate, hydroxyapatite, carbonate-apatite,

' chlorapatite, fluorapatite and mixtures thereof.

In US. Pat. No. 3,443,261 there is disclosed a method of formingstructures from a homogeneous mixture of a water-insoluble,micro-crystalline, ionizable, partial salt of collagen and calciumphosphate with or without inclusion of other ions such as found innaturally occurring bone and cartilage. The product consists primarilyof an intimate and homogeneous physical mixture of the variousingredients and various ions may be included to increase the hardness ofthe product and collagen cross-linking agents may be usedin'manufacturing the products so as to improve the moisture-resistanceand water-resistance of the products.

The principal purpose of the presentinvention is to provide a newcompositon of matter for the preparation of structures resemblingcartilage and bone tissue with improved moisture-resistant andwater-resistant properties. 7

The present invention contemplates formimg a new composition of mattercomprising a water-insoluble, colloidally dispersible, complex partialsalt of collagen with a polyvalent metal cation and an ionizable anion,withor without a homogeneous, intimate mixture of a salt of a polyvalentmetal and an ionizable anion in a mesoamorphous state.

The collagen molecule contains both carboxyl groups and amino groups,the free and available carboxyl groups being sites for the addition of acation and the free and available amino groups being sites for theaddition of ionizable anions. Both carboxyl and amino groups are alsopresent internally of the three intercoiled polypeptide chains butconditions of treatment are insufficient to penetrate the macromolecularstructure. Briefly, in forming the new composition, the collagen sourcematerial such as mechanically shredded and ground bovine collagen orother collagen source material is treated with a solution of apolyvalent metal hydroxide, the solution having a pH of from about 8 to12 preferably between 10.0 and 12. The polyvalent metals satisfactoryfor the purposes of this invention are those polyvalent metals whosehydroxides are soluble in water at pHs between pH 8 to pH 12 such as,for example, aluminum, cadium, calcium, chromium, iron, magnesium andzinc. The treatment of the collagen with the alkaline solution of themetal hydroxide results in a salt bridging reaction whereby the metalcation will react with free and available carboxyl groups of adjacentcollagen molecules to form a partial metal salt. This metal salt is thenpreferably recovered and washed free of soluble salts.

The recovered partial metal salt is treated with a dilute solution ofionizable acids such as hydrochloric acid, hydrobromic acid, hydroiodicacid, phosphoric acid, sulfuric acid, acetic acid, citric acid andoxalic acid. Other ionizable acids which will form amine salts with thefree and available amino groups on the collagen molecule aresatisfactory but from a practical commercial standpoint the foregoingacids are the most feasible. After reaction with the acid, the complexsalt may be recovered in dry form. Preferably, the reaction product isseparated by centrifuging to remove excess liquid and the product thenslurried in a water-miscible organic solvent such as methanol, ethanol,isopropanol and the like. The product is again centrifuged to separateit from the bulk of theliquid and then air dried or dried in an ovenpreferably under 100C. to form a dry, white, fluffy, fibrous material.

The fibrous material may be dispersed in water preferably by anattrition operation to produce a colloidally dispersible material andforms stable viscous gels at solids concentrations of as low as 0.5percent by weight. The dispersion preferably at higher concentrationsare dried to form desired shaped structures. The pH of the dispersionsand the specific drying method will determine the physicalcharacteristics of the products. For example, a product formed bytreating the collagen with a calcium hydroxide solution and subsequentlytreating the recovered calcium salt with phosphoric acid when dispersedin water in an amount of 3 grams of the calcium-phosphate-collagenreaction product in 497 grams of deionized water in a Waring Blendorexhibited an initial pH of 3.5 when slurried in the water and afterminutes attrition exhibited a pH of 3.7. The viscosity 2 hours afterpreparation of the dispersion measured with a Brookfield viscometerModel HAT at rpm. and C. was 12,200 cps. After 24 hours, the viscositywas 12,620 cps. and remained at this value for the period of test whichwas several days. A similar dispersion wherein the pH was adjusted bythe addition of dilute phosphoric acid to an initial pH of 2.9 exhibiteda final pH of 3.1. The viscosity after 2 hours was 14,680 cps. and after24 hours was 13,760 cps. and the viscosity again remained constant.

In both instances, the gel when poured into a shallow pan and allowed toair dry formed tough, flexible, amber colored cartilage-like structures.These same dispersions when freeze-dried formed porous, spongelikestructures, the interconnecting portions of material varying fromfiber-like to small filmor flake-like microscopic sub-structures.

The freeze-dried product formed from the gel as described above having apH of 3.7, exhibited a porous, sponge-like structure. When immersed inwater, the product exhibited a substantial swelling but retained itscoherent structure without disintegrating. Heat treatment as, forexample, heating the dry structure at 105C. for 3 hours reduces thedegree of swelling when immersed in water. On the other hand, when thepH of the dispersion is increased by the addition of ammonia to a pH ofabout 4.5 before freeze-drying, the product obtained shows a markedreduction in swelling. Air dried or oven dried products which arecontinuous films or non-porous structures exhibit these same waterabsorption and swelling characteristics when prepared from dispersionsat different pH values.

Ivory-like and bone-like structures are obtained by mixing with thedispersion of the partial salt of collagen a mesoamorphous salt of apolyvalent metal and the particular acid. In forming these structures,the amount of polyvalent metal salt may vary from several percent topercent based upon the weight of the partial salt of collagen. Thecompositions containing the lower proportions of metal salt will becartilage-like whereas those containing over about 40 percent of themetal salt will resemble ivory and bone. Obviously, the higher theproportion of polyvalent metal salt, the more dense the final productand there is no sharp division between the flexibility, pliability,density and hardness of the products within the stated range ofproportions there being a gradual decrease in flexibility and pliabilityand an increase in hardness as the proportion of the metal salt isincreased. The stability of these products will vary depending upon thepH of the dispersion from which the product is obtained in the manner asdescribed above.

In forming compositions for bone-like structures, other desired ions andthe so-called trace elements and radicals may be included in thedispersion or gel. For example, assuming a calcium-phosphate-collagensalt and calcium phosphate dispersion or gel, the fluoride, carbonateion, etc., may be included so as to form fluoro-apatite andcarbonate-calcium phosphate compounds such as thehydroxyapatite-carbonate compounds present in some bone tissues.

In the mammals, cartilage and bone must withstand one or more of severalforces such as compression, bending, twisting and impact forces. Theresistance to some of these forces and certain of the physical strengthcharacteristics of the products of this invention may be altered andimproved by incorporating in the aqueous dispersions or gels fiberswhich function as reinforcing elements.

The fibers may be formed of synthetic polymers such as, for example,polyesters, nylon, polytetrafluoroethylene, polyolefins andpolycarbonates, and of natural polymers such as, for example, collagenfibers, amylose fibers, chitin and the like. These fibers may be of anydesired size such as conventional textile sizes which vary from about Ito 10 deniers and vary in length from about one-fourth inch toconventional staple lengths of about I 9/l6 inch. The fibers may beincorporated as individual fibers or in some instances the fibers may befelted or woven or knitted into a desired porous or foraminous fabric insheet or tubular or other desired configuration which is subsequentlyimpregnated and coated with the calcium-phosphate-collagen salt andcalcium-phosphate dispersion. Fibers may constitute from about 1 percentto percent, preferably l5percent-60 percent, by weight, of the finishedproduct depending upon the specific application.

The complex partial calcium-phosphate-collagen salt is derived orprepared from undenatured collagen. The preferred source of collagen isbovine collagen or hide, that is, that portion of the hide which isessentially collagen and from which the hair and flesh have beenremoved.' Essentially, this material is also referred to as the coriumof the hide. Other sources of collagen such as,. for example, moosehide, pigskin, goatskin and sheepskin are also satisfactory. i

The collagen source material is preferably in a ground fibrous form suchas produced by the use of the conventional Urschel Mill. The finelydivided fibrous collagen is slurried in a solution of the metalhydroxide having a pH of between about 8 and 12. The solidsconcentration, that is, the proportion of fibrous collagen, may be asdesired, preferably up to about 10 percent, a 5 percent solids slurryconstituting a readily handleable slurry. It is, of'course,desirable toagitate the slurry constantly and it has been found that a convenientpractice includes starting the reaction with about 90 percent of themetal'hydroxide solution and slowly adding additional solution so as tomaintain the desired pH. In general, a mixing period of from about l0minutes to about 30 minutes depending upon the solids concentration issatisfactory. At a 50 percent solids concentration a period ofIS'minutes is satisfactory.

The temperature" is maintained below about 30C. and

should be below a temperature at which collagen is deaqueous acidsolution ispreferably premixed with the water-miscible organic solventto avoid the possibility of contacting portions of the metal-collagenreaction product with more concentrated acid than other portions of themetal-collagen reaction product. The amount of acid present may varyfrom about 0.2 to about 0.8 millimole of acid per gram of collagen.Bovine collagen contains about 0.78 millimole of available primary aminogroups per gram of collagen and products prepared from such collagenwill have a bound acid content ranging from about 0.15 to about 0.7millimole per gram of collagen (about percent to about 90 percent of thestoichiometric bound acid content) with an average of'about- 0.58millimole per gramof collagen.

The reaction may be carried .out in a planetary mixer so as to insure athorough dispersion of the metalcollagen salt in the dilute acidsolution. The solids concentration may vary up to about 15 percent andthe period of mixing may range from about 10 to 30 minutes. Asatisfactory mixing may be performed in a conventional Hobart mixer at-asolids concentration of about 10 percent'for about 15 minutes. Becauseof the nature of the slurried collagenous material, concentrations arelimited because of handling difficulties. The specific type of mixingapparatus utilized in both reaction operations may be selected on thebasis of the solids concentration. Simple paddle mixers or stirrers aresatisfactory for the lower concentrations while planetary mixers andapparatus such as the Cowles Dissolver and a Bauer mill may be used forthe higher solids concen trations.

Following the reaction with the acid, the complex partial ionizable saltis separated from the bulk of the liquid as by centrifuging and the saltor reaction product recovered asa wet cake. The wet cake is slurried inwater or a water-miscible organic solvent, preferably in an organicsolvent, to remove soluble substances and the salt separated from thebulk of the liquid and then dried to recover the complex salt.Preferably, the water-miscible organic solvent is utilized at least inthe final washing step to provide a dry product which is fluffy andfibrous in nature. The replacement of water by an organic solventreduces swelling and also pre vents hydrogen bonding during drying.

The dry, white, fluffy product is dispersible in water and preferablythe dispersion is formed by the use of equipment capable of attritingthe complex salt to a colloidal condition. Dispersions containing 0.5percent solids exhibit a decrease or increase in viscosity of not morethan about 10 percent within 24 hours after preparation. Thereafter, theviscosity remains substantially constant, that is, it does not changemore than a few percent over a storage period of 1 week when stored at5C. under conditions which prevent the loss of water.

In preparing the structure from the dry, white, fluffy product dispersedin the aqueous medium, the character of the structure will be determinedby the specific drying method. A dispersion free of dispersed gas whenair dried will produce a hard, dense, horn-like structure. Where thedispersion contains dispersed gas, the structure will be a porous buthard, horn-like structure. A dispersion free of dispersed gas whenfreeze dried will produce a structure having porosity but is hard andrigid comparable to the natural cancellous bone structure. Where thestructure is freeze dried from a dispersion containing dispersed gas as,for example a foamlike dispersion, the structure will be highly porous.If desired, a finely divided blowing agent may be incorporated in thehigh solids dispersions or mixtures before drying. Blowing agents, suchas for example ammonium carbonate, upon heating of the mixture becomedecomposed to gases thereby creating pores, the size of which may bevaried by varying the particle size of the blowing agent.

In order to simplify the description, reference will be made to thepreparation of structures comparable to natural bone tissue by referenceto the use of the calciumphosphate-collagen complex salt and calciumphosphate. It is obvious that where his desired to use other cations andother anions they may be substituted fo the calcium and phosphate ionsto form structures with similar properties.

In forming synthetic cartilage-like and bone-like structures of thepresent invention, mesoamorphous calcium phosphate is mixed with anaqueous gel of the water-insoluble ionizable complex collagen salt.Since bone tissues contain some citric acid and phosphate salts, thecomplex collagen salt is preferably prepared byv treatment of thecollagen source material with citric acid or phosphoric acid or an acidphosphate salt so as to avoid the presence of what may be termed foreignions in the product. The calcium phosphate may be formed by mixingsolutions of a soluble phosphate, such as, for example, sodium phosphateand a soluble calcium salt,such as, for example, calcium acetate, so asto provide a desired molar ratio of calcium to phos phate, preferably toprovide a ratio of about 1.6:1.

In the biological formation of bone tissue and particularly the hardtissue of teeth, an apatite-carbonate compound is produced whichapparently accounts for the extreme hardness of such tissue. In theproduction of the structures according to this invention, desiredamounts of the various anions may be incorporated in the gel mixtures,preferably in the preparation of the calcium phosphate, to providerelative ratios approximating those of natural bone tissue. In theaddition of, for example, the carbonate anion and the fluoride anion,about 1 to 10 molar percent, preferably 2 to percent, of the phosphateanion may be replaced by the carbonate anion and about 0.05 to 2 molarpercent, preferably 0.1 to 0.6 percent, of thephosphate ion may bereplaced by the fluoride anion. Where it is desired to incorporate otherions such as the'carbonate and fluoride anions, solutions of salts suchas sodium carbonate ane sodium fluoride are preferably added during thepreparation of the calcium phosphate. Preferably, these solutions areadded to the soluble phosphate solution so that upon mixing with thesolution of the calcium salt, the salts containing the other ions willcoprecipitate with the calcium phosphate.

The solutions of the soluble salts are preferably mixed under vigorousagitation. The resulting slurry of the insoluble salts will generallyhave an alkaline pH of from 10.5 to about 12. The mesoamorphous calciumphosphate is separated by filtration and washed thoroughly with water soas to remove soluble salts. The recovered wet salt is slurried in waterand just prior to the addition of the slurry to an aqueous gel of themicrocrystalline collagen salt, the pH of the slurry is preferablyadjusted to a pH of between about 3 to 5 by the addition of acetic acid.Alternatively, the slurry having the alkaline pH is slowly added to thecomplex collagen salt dispersion which has a pH of about 2.5 to about5.5 with constant agitation. The pH of the combined slurry anddispersion is then lowered to a value within the pH range of thedispersion by the addition of an acid such as acetic or phosphoricacids. As the pH of the liquid containing the slurried calcium phosphateis lowered to an acid pH, there results a structural crystallinetransformation of the calcium phosphate.

The mixtures are dried to a moisture or water content of from about 5percent to about 25 .percent. Where porous structures are desired,drying is preferably effected by a freeze drying process. After themoisture content has been reduced to a value within this range, thestructures are preferably heated, as in an oven in an inert atmosphere,to a temperature between about 100C. and about 120C. for from 2 tohours. This heating step further increases substantially the stabilityof the structural characteristics of the products in the presence ofaqueous liquids.

The predetermined structure is formed by extrusion or molding or otherdesired techniques. To form relatively thin structures, the combinedslurry and dispersion may be placed in a tray to a desired thickness anddried by any desired method. Alternatively, heavy dispersions orpaste-like dispersions (higher solids concentration dispersions) may becast in desired molds. As a further alternative, blocks may be formedand desired products formed by machining.

The following specific examples are set forth merely to illustrate theapplication of the present invention:

EXAMPLE 1 Urschel milled, vacuum freeze dried bovine collagen was mixedwith water to form a swollen fibrous mass containing 30 percent solids.A solution of calcium hydroxide was prepared in deionized water, thesolution having a pH of l 1.6. 670 parts by weight of the swollenfibrous mass was mixed into 3,330 parts of the calcium hydroxidesolution. Approximately 300 ml. of l N calcium hydroxide solution wasadded during the mixing operation to maintain the pH at l 1.6. Themixing operation was carried out for 15 minutes at room temperature. Theslurry was then centrifuged to a 30 percent solids concentration wetcake. A 10 percent solids slurry was prepared in a Hobart mixer byadding 670 parts of the wet cake to a preformed mixture of 1330 parts ofisopropanol and 13.34 parts of an percent solution of phosphoric acid.Mixing was continued for 15 minutes. The resulting reaction product wasseparated from the bulk of the liquid by centrifuging to a solidscontent of about 30 percent. The recovered wet material was slurried in1,330 parts of isopropanol, again centrifuged and then air dried.

The calcium-phosphate-collagen complex salt thus prepared was a white,fluffy, fibrous material. Upon forming a 3 percent solids dispersion ofthe product in deionized water in a Waring Blendor for 15 minutes thereresulted a viscous dispersoid having a viscosity 2 hours afterpreparation of 12,200 cps. as measured in a Brookfield Viscometer, ModelHAT, at 10 rpm. and 25C. After 24 hours, the viscosity had risen to12,620 cps. and remained practically constant during further storage at5C. At the initial stage of the dispersing operation, the pH of theliquid was about 3.5 and at completion the pH was about 3.7.

In a like manner, another dispersoid was prepared and the initial pH wasadjusted to 2.9 by the addition of dilute phosphoric acid. The final pHwas 3.1. The viscosity 2 hours after preparation was 14,680 cps. anddecreased to 13,760 cps. at the end of 24 hours. Further storageexhibited no significant change in viscosity.

A portion of each dispersoid was poured into a shallow pan to a depth ofabout one-half inch and air dried to a moisture content of about 10%after which the pans were transferred to an oven maintained at C. Thefilm-like structure was dried to a moisture content of about 3 percent.

A portion of the dispersoid having a final pH of 3.7 was freeze driedfor 12 hours to a water content of about 4 percent (40 to 50C., vacuum 5microns, heating cycle not exceeding 30C. with condensation of water at60C.). The pH of another portion of the dispersoid was raised to about apH of 4.5 by the addition of a calcium hydroxide solution. Thedispersoid was then freeze dried.

Both products had a porous, sponge-like structure with fine pores. Theinterconnected solid material examined under a microscope appeared tohave a fibrous and flake-like structure without visible evidence ofseparated calcium phosphate. Both structure when placed in water exhibita swelling but remain as coherent, nongelatinous bodies and will notdisintegrate. The higher the pH of the dispersoid prior to freeze dryingthe lower the swelling.

EXAMPLE 2 A calcium-phosphate-collagen complex salt was prepared asdescribed in Example 1.

A calcium phosphate slurry was formed by mixing a solution of 4.78 partsof trisodium phosphate in 100 parts by weight of deionized water with asolution of 17.6 parts of calcium acetate in 100 parts of deionizedwater. The resulting slurry had a pH of 11.3. The calcium phosphate wasrecovered and washed with deionized water to remove soluble salts. Adispersoid was formed containing 6% by weight thecalcium-phosphate-collagen complex salt in deion- I ized water. Thedispersoid in this instance had a pH of 4.5. The freshly recoveredcalcium phosphate wet cake (about 40% solids) in an amount equivalent toapproximately 2.5 times the quantity of collagen complex salt was addedto the dispersoid in a Sigma'blade mixer. The pH of the wet cake wasabout 10.5. The pH' of the mixture was slowly adjusted to a pH of about4.5 to 5.0 by the addition of phosphoric acid. Mixing was continued forabout 2 hours and the temperature was maintained below 25C. duringadjustment of the pH (the first one-half hour) and the temperature thenallowed to increase to about 80C. by the end of the mixing operation.The final mixture was in the form of a wet,

moldable mass A portion of the mass was molded into a rectangularstructure and allowed to air dry at room'temperature with forced aircirculation. The air dried material containing about moisture was thenoven dried a 105C. for about 12 hours.

The product was hard, dense bone-like in character. A like hard,bone-like but lower density productwas obtained when a like rectangularmoldedv structure was freeze dried as described in Example I. I

The products of both Examples 1 and'2 have a sufficiently highstructural stabilityto withstand boiling in water withoutdisintegrating. Samples of products 'of Examples 1 and 2withstand-boiling water for a period of at least 6 hours without loss ofcoherence in the structure.

In the 'event other anions are desired, such as, for example, thefluoride or carbonate ions, suitable salts such as sodium or calciumfluoride or sodium carbonate are incorporated in the solutions duringthe forma tion of the calcium phosphate.

The foregoing examples are'merely illustrative. For example, inpreparing the collagen complex salt, other metal hydroxides aresubstituted for the calcium hydroxide in Example 1. It is recognizedthat certain hydroxides such as aluminum and chromium hydroxides areprobably not considered as being in true solution but exist at the highpl-ls in such state that they will react as does the calcium hydroxide.Thus, in speaking of metal hydroxides soluble at pH 8 to pH 12, it isintended that these. types of metal hydroxides are included in thedefinition. The physical properties of the products formed by suchsubstitution are substantially the same as described in Examples 1 and2.

Any of the described acids may be substituted for the phosphoric acid ofExample 1 in preparing the collagen complex salt. For example, citricacid may be used and the added salt of Example 2 may be calciumphosphate. Alternatively, mixtures of the acid may be used.

As also described hereinbefo're, the structural characteristics may bealtered by incorporating in the dispersoid inert fibers. For example,the bend or twisting strengths of the products of Examples 1 and 2 areincreased by incorporating l denier nylon 66 staple fibers or otherfibers. Urschel milled collagen fibers of a diameter of 35-40 micronsand having lengths of about one-fourth inch are also satisfactory.

The compositions as disclosed herein and particularly those having thehigher ratios of calcium phosphate compounds to collagen aresatisfactory for the production of three-dimensional, self-supporting,impact-resistant structures. The three dimensional structures arereadily machined, sawed, drilled and worked and may be given a fairlyhigh polish. The hard, dense, compact structures when machined andpolished, for example, have much the same appearance and feel as ivory.Accordingly, the structural material may be used to replace ivory inmusical instruments such as piano keys. Billiard balls, costume jewelryand blocks may also be used for sculpturing. Because of the collagencontent, a variety of dyes may be used to provide any desired color.

As indicated hereinabove, the principal inorganic constituent of thecompositions, that is, the calcium phosphate compounds may vary in theratio of calcium to the phosphate ion from about 1:1 to about 1.6:1 andit is probable that in some instances there occurs in the mass a mixtureof specific calcium phosphate compounds. In the production of articlessuch as, for example, combs, spatulas, etc., a high solids contentaqueous mixture of the dispersoid and clacium phosphate may be utilizedwith added fibers to increase flexural strength and a cross-linkingagent to improve moisture and water resistance. The mixture may beshaped by the use of press molds of the desired configuration.

In preparing the compositions for use in bone surgery where thestructure will be inserted in a mammal such as a dog or cat,bactericides, fungicides and antibiotics may be incorporated in theproducts either during their preparation or, since many of the productsare porous, these substances may be introduced by impregnationprocedures. Hemostats may be included for a specific purpose whereeither a coagulant or anti-coagulant is desired in a specific site.Alternatively, a high solids content aqueous mixture of the dispersedcollagen salt and calcium phosphate or a wet mass of a ground-up driedproduct may be used in the treatment of bone damage in the manner inwhich ground-up, moist bone is now used. In this instance, thebactericide, fungicide, antibiotic, hemostat or other desired additiveis incorporated in the moist massfExamples of agents which may beincluded are well known to those skilled in the art and include suchsubstances as chlortetracycline, erythomycin, bacitracin and heparinsodium, etc.

As disclosed in U. S. Pat. No. 3,443,261, the collagenous constituent ofthe bone-like structures is a waterinsoluble, ionizable, partial salt ofcollagen such as a phosphoric acid or citric acid partial salt whereinthe salt has a bound acid content of from about 50% to of thetheoretical stoichiometric bound acid content. The collagenousconstituent of the structures of the present invention contains a boundpolyvalent metal and a bound acid content of from about 20% to about 90%of the theoretical stoichiometric bound acid content. The partial saltof collagen as utilized in U. S Pat. No. 3,443,261 can not be convertedto the collagenous material utilized in the present invention bytreatment with an alkaline material such as barium hydroxide or basicaluminum acetate. Treatment with such alkaline materials removes thebound acid. The resistance of these various collagenous materials toboiling water differs markedly.

The differences in the resistance to boiling water may be illustrated bythe following example:

EXAMPLE 3 A water-soluble, ionizable, partial salt of collagen wasprepared in accordance with Example 1 of U. S. Pat. No. 3,443,261. To anaqueous solution of hydrochloric acid having a pH of 2.4 there was addedsufficient Urschel milled, vacuum freeze dried bovine collagen so as toform a slurry containing 1% by weight of collagen. After slurrying atroom temperature for about 15 minutes, the mass was transferred to aWaring Blendor where it was subjected to attrition for about 25 minutesat a temperature which was 'not allowed to exceed 25C. The resulting gelhad a pH of about 3.6. The gel was spread in a pan to a depth of aboutone-half inch and freeze driedover night (40 to 50C., vacuum 5 microns,heating cycle not exceeding 30C. with condensation of sublimed water at60C.). The resulting porous, sponge-like product was identified asSample A.

A portion of Sample A was placed in a 2 percent aqueous solution ofbarium hydroxide having a temperature of about 30C. The sample wasstirred in the solution for about minutes, washed with a 50/50 by volumesolution of ethanol and water and finally with ethanol. The sample wasthen dried in an air oven at a temperature of about 95C. The driedsample was identified as Sample B.

Another portion of Sample A was treated in the same manner with a 2%solution of barium hydroxide in a 50/50 by volume solution of ethanoland water. The dried sample was identified as Sample C.

A white, fluffy, fibrous calcium-phosphate-collagen complex salt wasprepared as described in Example 1. Sufficient of the fibrous materialwas added to deionized water in a Waring Blendor to form a 1% by weightdispersion and subjected to attrition for 15 minutes, maintaining thetemperature below 30C. The pH of the resulting dispersion had a pH ofabout 3.6. The gel was spread in a pan to a depth of about one-half inchand freeze dried overnight under conditions as set forth above. Theresulting porous, sponge-like product was identified as Sample D.

In the same manner as one-half percent by weight dispersion was formedand spread in a pan to a depth of about one-half inch and freeze driedover night under conditions as set forth above. The resulting porous,sponge-like product was identified as Sample E.

A sixth sample was identified as Sample F This sample was cut from asample which had been prepared prior to May 1969 as described for thepreparation of Sample D but after drying had been placed in distilledwater and maintained in a closed container since that time.

All samples in dry condition exhibited a withe, porous, fibrous,sponge-like appearance, Sample E being somewhat more porous and lower indensity than the other samples.

Portions of each of the samples when placed in water at room temperatureexhibited a swelling to about a two-fold increase in thickness afterbecoming wet throughout. Samples A, D, E and F exhibited no furtherchange in appearance after several hours, however, Samples B and Cexhibited some internal separation and individual fibers appeared alongthe edges of the samples.

Portions of each of the samples when placed in boiling water exhibited asimilar swelling after becoming wet throughout. The subsequent action ofthe samples is set forth in the following table:

Sample B Sample C Sample D) Sample E) Sample F) I claim:

1. The method of preparing a three-dimensional, water-insolublestructure comprising reacting undenatured collagen with a solution of apolyvalent metal hydroxide having a pH of between about 8 and 12 and ata temperature not exceeding 30C., the polyvalent metal hydroxide beingsoluble in water at a pH of between about 8 and 12, recovering themetal-collagen reaction product, reacting the metal-collagen reactionproduct with from about 0.2 to about 0.8 millimole of an ionizable acidper gram of collagen, based upon collagen containing about 0.78millimole of available primary amino groups per gram of collagen to forma complex, partial salt of collagen with the polyvalent metal cation andthe ionizable anion containing from about 0.15 to about 0.7 millimole ofbound acid per gram of collagen, based upon collagen containing about0.78 millimole of available primary amino groups per gram of collagen,shaping the complex partial salt into a predetermined structuralconfiguration and drying the shaped structure to a moisture content offrom about 5 percent to 25 percent, heating the dried structure to atemperature between about C. and C. in an inert atmosphere andmaintaining the structure at such temperature for from 2 to 10 hours.

2.'The method of claim 1 wherein the polyvalent metal hydroxide iscalcium hydroxide.

3. The method of claim 1 wherein the acid is phosphoric acid.

4. The method of claim 1 wherein the polyvalent metal hydroxide iscalcium hydroxide and the acid is phosphoric acid.

5. The method of claim 1 wherein calcium phosphate is added to thecomplex partial salt of collagen before shaping into the predeterminedstructural configuration.

6. A unitary three-dimensional structure comprising a water-insoluble,complex partial salt of collagen con taining a bound polyvalent metalion, the polyvalent metal being one whose hydroxide is soluble in waterat a pH between about 8 and 12, and containing a bound ionizable acidanion, the bound polyvalent metal ion replacing at least some of thefree and available carboxyl groups of the collagen, the partial saltcontaining from about 0.15 to about 0.7 millimole of bound acid per gramof collagen based upon collagen containing about 0.78 millimole ofavailable primary amino groups per gram of collagen, and the structurebeing further characterized by withstanding immersion in boiling waterfor at least about 6 hours.

7. A unitary three-dimensional structure as defined in claim 6 whereinthe polyvalent metal ion is calcium.

10. A unitary three-dimensional structure as defined in claim 6 whichincludes intermixed calcium phosphate.

UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION Patent No. 3,7 7,87 I Dated Ootober 23, 1973 In t fl Mamgrtg M. Cruz Jr.

It is certified that error appears in the above-identified patent andthat said Letters Patent are hereby corrected as shown below:

Column 1, line 5, "magnesium fluorine," should read --magnesium,fluorine- Title Page 1 and Column 1, line 1 "Phosthetic" should read"Prosthetic".

Column 11, line 5 4, "withe" should read --white-.

Signed and sealed this 15th day of April 75.

(321.1.) Attest:

C. RAE- SHALL DANN RUTH. C. EZASOI Commissioner of Patents attestingOfficer an Trademarks FORM PC (149) USCOMM-DC 60376-F'69 IL. OVIIIIIIIIT"IIII'IIG OFFICE Ill! O-li*334

2. The method of claim 1 wherein the polyvalent metal hydroxide iscalcium hydroxide.
 3. The method of claim 1 wherein the acid isphosphoric acid.
 4. The method of claim 1 wherein the polyvalent metalhydroxide is calcium hydroxide and the acid is phosphoric acid.
 5. Themethod of claim 1 wherein calcium phosphate is added to the complexpartial salt of collagen before shaping into the predeterminedstructural configuration.
 6. A unitary three-dimensional structurecomprising a water-insoluble, complex partial salt of collagencontaining a bound polyvalent metal ion, the polyvalent metal being onewhose hydroxide is soluble in water at a pH between about 8 and 12, andcontaining a bound ionizable acid anion, the bound polyvalent metal ionreplacing at least some of the free and available carboxyl groups of thecollagen, the partial salt containing from about 0.15 to about 0.7millimole of bound acid per gram of collagen based upon collagencontaining about 0.78 millimole of available primary amino groups pergram of collagen, and the structure being further characterized bywithstanding immersion in boiling water for at least about 6 hours.
 7. Aunitary three-dimensional structure as defined in claim 6 wherein thepolyvalent metal ion is calcium.
 8. A unitary three-dimensionalstructure as defined in claim 6 wherein the anion is the phosphate ion.9. A unitary three-dimensional structure as defined in claim 6 whereinthe polyvalent metal ion is calcium and the anion is the phosphate ion.10. A unitary three-dimensional structure aS defined in claim 6 whichincludes intermixed calcium phosphate.